I have underlined the CpG that I identified and I have a question abou it (I'm probably a little confused and would greatly appreciate your assitance):If I understand correctly I am supposed to perform two PCR reactions, one with methylated primers (forward and reverse) and the other with unmethylated primers (also forward and reverse). Now, if my DNA sample is methylated then the methylated set of primers will amplify it, since the CG sequences remain unchanged after bisulfite teratment, but what if the sample is unmethylated? In this case, the bisulfite treatment will change the CG in the original sequence into 5'UG3'. From here, in order to receive a strand that my unmethylated forward primer is complementary to I need to duplicate this strand using the unmethylated reverse primer. And this is where I get confused: Why does the unmethylated reverse primer not contain any CG sequences? Wouldn't it cause amplification (of one strand) regardless of methylation status? And if it did contain CG sequences they would be in the TG form like in the unmethylated forward primer and then we wouldn't get amplification at all because we nee the first duplication of the strand to turn 5'UG3' into 5'CA3' that is complementary to the 5'TG3' of the primer. How does this work and why does the unmethylated reverse primer not contain any CG? Sorry for the length, if anyone could clarify this for me I would be forever thankful. really

-Adelle-

Hi Adelle,

you are almost there, there are CG sequences in the form of CA, because it's the complement strand (or another words the complement of the same strand that was amplified with the forward strand).

Just remember with bisulfite PCR, it is strand specific, so the primer pairs only target one strand of DNA because after conversion, the strands are non complementary.

Nick

-methylnick-

QUOTE (methylnick @ May 5 2007, 10:07 AM)

Hi Adelle,

you are almost there, there are CG sequences in the form of CA, because it's the complement strand (or another words the complement of the same strand that was amplified with the forward strand).

Just remember with bisulfite PCR, it is strand specific, so the primer pairs only target one strand of DNA because after conversion, the strands are non complementary.

Nick

Thank you so much for making it clear.As promissed I'm forever grateful